PRO-APOPTOTIC GENE EXPRESSION COMPARISON BETWEEN RECOMBINANT DISINTEGRINS R-MOJ-DA, R-MOJ-DN, AND R-MOJ-DM-TREATED SK-MEL-28 CELLS
Andrew U, Julio Soto.
San José State University, San José, CA.
Disintegins are small, non-enzymatic peptides that bind and activate integrins. Integrins can initiate signal transduction pathways leading to cell migration, survival, and apoptosis. Previous studies have shown that three mutated recombinant mojastin peptides (r-Moj-DM, r-Moj-DN, and r-Moj-DL) induce apoptosis of SK-Mel-28 cells. Three other recombinant mutants with a second aspartate, carboxyl to the RGD (r-Moj-DA, r-Moj-DG, and r-Moj-DV) failed to induce apoptosis of treated SK-Mel-28 cells. Additionally, RNA sequencing data of SK-Mel-28 cells treated after 6 hr with r-Moj-DM showed significant upregulation and downregulation of 40 genes. In the present study, we compared the expression of several pro-apoptotic genes after treatment with two apoptotic-inducing peptides (r-Moj-DM and r-Moj-DN) and one non-apoptotic peptide (r-Moj-DA). We hypothesized that gene expression of seven pro-apoptotic genes will be significantly higher in r-Moj-DM and r-Moj-DN treated cells. SK-Mel-28 cells were treated for 1, 2, 4, and 6 hrs with either 5 µM of r-Moj-DA, r-Moj-DM, or r-Moj-DN peptides. Following treatment, mRNA was isolated from treated cells and cDNA synthesized using a Qiagen Reverse Transcriptase Kit. Quantitative (Q) PCR was used to examine gene expression levels and compared to expression levels of untreated cells. Our results from r-Moj-DA-treated cells indicate that expression levels of the seven pro-apoptotic genes tested showed a non-significant upregulation as compared to the expression of the same genes from r-Moj-DM-treated cells. In the next weeks, we will be examining the expression of the same seven pro-apoptotic genes in r-Moj-DN-treated cells.